RNA SYNTHESIS IN VITRO

Talib, S., and J.E. Hearst. 1983. Initiation of RNA synthesis in vitro by vesicular stomatitis virus Single internal initiation in the presence of aurintricarboxylic acid and vanadyl ribonucleoside complexes. Nucleic Acids Res 11 7031. 

Additional experiments characterizing SL RNA transcription showed that 3 end formation (termination) of SL RNA synthesis in vitro was also unusual in that it... 

The ribose equivalent 5-ethynylcytidine (EC) proved to be a valuable tool for monitoring RNA synthesis in vitro and in vivo (mice), exhibiting similar sensitivity to EU and was faster metabolized than EU [8]. Interestingly, RNA labeling with the purine nucleoside 5-ethynyl-adenosine (EA) in contrast did result in unspecific labeling of both cytoplasm and nuclei, and is not suitable for the specific detection of RNA synthesis in cells [8]. 

Three steps can be distinguished in the RNA synthesis in vitro. They are (1) the binding of RNA polymerase to DNA (2) the stabilization of the complex, accompanied by the fixation of the first nucleotide of the RNA chain (a purine nucleotide) (3) the elongation of the RNA chain. 

Week and Wagner (1978) compared transcription of isolated nuclei from VSV-infected and uninfected MPC-11 cells to obtain a more direct measurement of nuclear polymerase activity by the method of Smith and Huang (1976). At 2 hr postinfection, they found approximately a 50% decline in the rate of RNA synthesis in vitro by nuclei isolated from infected cells. The toxin a-amanatin was used at a concentration of 1 xg/ml to distinguish the RNA polymerase II activity from that of the more resistant RNA polymerases I and III. During the first hour after infection, there was a rapid loss in the activities of all three RNA polymerases. Subsequently, the level of RNA polymerase II activity continued to decline until 4 hr postinfection while the level of combined RNA polymerase I and III activity remained constant. 

Acetylation of histones reduces their ability to inhibit RNA synthesis in vitro (Allfrey et al., 1964). The acetylation rate and concentration of acetyl-group histones are higher in transcriptionally active chromatin. Deacetylation of histones, at the same time, does not slow down. The rate of histone acetylation increases in types of cell where RNA synthesis is stimulated, i.e., spleen cells treated with eryth-ropoetin, liver cells under the influence of cortisol, milk gland cells treated with insulin, etc. (Allfrey et al, 1972). Inhibition of nuclear activity is accompanied by the deacetylation of histones. 

As the results of molecular hybridization and determination of base sequence have shown, RNA formed during phage reproduction in vivo on the basis of the circular double-helical replicative form of DNA (Hayashi et al., 1964) is complementary to only one of the two DNA chains, i.e., to that which is complementary, in turn, to the single-stranded DNA of the mature phage. In vitro when the circular structure of DNA is disturbed, both strands of replicative DNA can act as substrate for RNA-polymerase. The single-stranded DNA of this phage can also act as template for RNA synthesis in vitro, with the preliminary formation of DNA/ RNA hybrids (Chamberlin and Berg, 1964). 

Meanwhile, if synthesized in vitro, complementary RNA forms secondary double helices (Geiduschek et aL, 1962). Later work in the same laboratory (Geiduschek et al., 1964) showed that RNA synthesis in vitro (on DNA templates) is symmetrical (i.e., it copies both DNA strands) if the DNA is partially degraded. If the DNA molecules are intact, RNA synthesis is asymmetrical, i.e., in this case only one strand acts as template. This evidently accounts for the results obtained by Bresler and co-workers (1964), who obtained artificial double-helical DNA hybrids, introduced them intoB. cells as transforming agent, and found that both... 

Wu, A. M., Gosh, S., Willard, M., Davison, J., Echols, H. Negative regulation by lambda Repression of lambda RNA synthesis in vitro and host enzyme synthesis in vivo. In A. D. Hershey (editor). The bacteriophage lambda, p. 589-598. Cold Spring Harbor Laboratory 1971. 

ARCAs are incorporated into RNA exclusively in the correct orientation to an extent that is similar to the standard cap (see previously), which makes them potentially useful compounds in terms of increasing translational efficiency when incorporated into RNA. Similarly, they should be effective for inhibiting protein synthesis as free analogs. To test the influence of the ARCAs on protein synthesis in vitro, we use the microccocal nuclease treated rabbit reticulocyte lysate system (RRL system) optimized for cap-dependent translation (Cai et al., 1999). Highly cap-dependent translation is achieved at 100 mM potassium acetate and 1.4 mM magnesium chloride. 

These observations are consistent with the conclusion that auxin treatment leads to the synthesis of BS cellulase, which then accumulates in smooth ER vesicles. There is direct evidence that the synthesis occurs in rough ER vesicles (11). Cellulase activity was shown (25) to increase in RNA-rich pea microsomes, provided these were isolated from auxin-treated tissue, when the preparations were incubated with ingredients necessary for carrying out protein synthesis in vitro. Messenger RNA (mRNA) from these microsomes has been translated in a different ribo-somal system and shown to synthesize BS cellulase protein (II). Thus, it is legitimate to use the term "induction to apply to the ability of auxin to evoke the appearance of mRNA for BS cellulase. 

Vol. XXX [61], DNA- and RNA-Directed Synthesis in Vitro of Phage Enzymes. P. Herriich and M. Schweiger. 

Inhibition of cell growth and DNA, RNA, and protein synthesis in vitro by fentanyl, sufentanil, and opiate analgesics Nassiri, M. Reza Flynn, Gordon L. Shipman, Charles, Jr. 

Telomere length is maintained by telomerase, an enzyme composed of one polypeptide subunit and one RNA subunit. The catalytic subunit of telomerase bears homology to retroviral reverse transcriptases, consistent with its role in catalyzing synthesis of DNA on an RNA template. The 3 end of a telomeric G-rich overhang is the primer for addition of new telomeric sequence, which is templated by the complementary sequence in the telomerase RNA subunit. In vitro, G-rich telomeric sequences readily form G-quadruplexes with distinctive structural features determined by telomere sequence and strand orientation. G4 DNA formation could in principle promote telomere telomere interaction [Figure 4(A)] protect the 3 end from extension or nucleolytic attack [Figure 4(B)] or stabilize t-loops [Figure 4(C)]. 

Prokaryotic RNA polymerase - A single RNA polymerase catalyzes the synthesis of all three E. coli RNA classes—mRNA, rRNA, and tRNA. This was shown in experiments with rifampicin (Figure 26.4a), an antibiotic that inhibits RNA polymerase in vitro and blocks the synthesis of mRNA, rRNA, and tRNA in vivo. 

It has been shown, in vitro, that proflavine (30 /xM) inhibits bacterial DNA polymerase by 85% and RNA polymerase by 30% (Hurwitz etaL, 1962). Some typical data for the RNA system are shown in Fig. 10.4. The inset graph (of the reciprocal of the velocity plotted against the reciprocal of the DNA-primer concentration) points to the site of action of proflavine as being mainly on this primer, thus inhibiting DNA and RNA synthesis in the living bacterium. The intercalating mechanism by which aminoacridines combine with DNA helices and impede strand separation is outlined in the next Section (10.3.2). 

The proposals to use SGe analogs as antitumor agents were based on evidence that these compounds suppress protein, RNA, and DNA synthesis in vitro [6], At low concentrations, germanium blocks cellular proliferation, resulting in reproductive death. At higher concentrations, cell death is a result of cytolysis [39]. 

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